A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased
heparin affinity was shown to have normal inhibitory activity which d
ecreased slowly at 4 degrees C and rapidly at 41 degrees C. On electro
phoresis the freshly isolated variant had an anodal shift relative to
native antithrombin due to the mutation. A further anodal transition o
ccurred after either prolonged storage at 4 degrees C or incubation at
41 degrees C due to the formation of a new inactive uncleaved compone
nt with properties characteristic of L-form (latent) antithrombin. At
the same time, polymerization also occurred with a predominance of di-
, tri-, and tetra-mers. These findings fit with the observed mutation
of the conserved asparagine (187) in the F-helix destabilizing the und
erlying A-sheet of the molecule. Evidence of A-sheet perturbation is p
rovided by the increased rate of peptide insertion into the A-sheet an
d by the decreased vulnerability of the reactive loop to proteolysis.
The spontaneous formation of both L-antithrombin and polymers is consi
stent with our crystal structure of intact antithrombin where L-form a
nd active antithrombin are linked together as dimers. The nature of th
is linkage favors a mechanism of polymerization whereby the opening of
the A-sheet, to give incorporation of the reactive center loop, is ac
companied by the bonding of the loop of one molecule to the C-sheet of
the next. The accelerated lability of antithrombin Rouen-VI at 41 ver
sus 37 degrees C provides an explanation for the clinical observation
that episodes of thrombosis were preceded by unrelated pyrexias.